This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of human Natural Killer Cell Activating Factor II, sometimes hereinafter referred to as xe2x80x9cNKAF IIxe2x80x9d.
The polypeptide of the present invention is a member of the natural killer cell activating factor (NKAF) family and shows homology to NKAF and human eosinophil; major basic protein. Natural killer cells (NK cells) show a destructive effect on specific cancer cells. Lymphokines affecting the activity of these NK cells have therefore attracted attention. It is reported that interleukin-2 and interferon enhance the activity of NK cells (Herberman, R. B., et al., Immunol. Rev., 44:13 (1979); Vose, B. M., et al., J. Immunol. 130:768 (1983) and Domzig, W. etal., J. Immunol., 130:1970(1983)).
NK cells are proposed to function as natural surveillance to deter cancer development in the body (Whiteside, T. and Herberman, R. B., Clin. Immunol. Immunopathol 58:1-23 (1989) and Trinchieri, G., Adv. Immunol 47:187-376 (1989)). NK cells are also important in controlling viral infection and the regulation of hematopoiesis (Trichieri, G., Adv. Immunol., 47:187-376 (1989); Kiessling, R., et al., Eur. J. Immunol. 7:655-663 (1977)).
The following facts indicate that NK cells play important roles in the host defense against cancer. Namely, a nude mouse lacking T cells by having a high NK activity does not always suffer from spontaneous or chemically induced carcinogenesis at a high frequency (Rygaard, J. et al., Immunol. Rev., 28:43 (1975); Stutman, D., et al., Science, 183:534 (1974)); and the metastasis of transplanted cancer cells is promoted in a beige mouse having T cells but a genetically low NK activity (Shimamura, K. and Tamaoki, K., Jikken Igaku, 2:398 (1984); James E. Talmadge, et al., Nature, 284:622 (1980)) and a mouse having an artificially lowered NK activity (Shimamura, supra).
Human eosinophil major basic protein (MBP) has a nearly identical sequence to that of known natural killer cell activating factor I. Human MBP is one of the principal mediators of injury to parasites in tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil activation. MBP has no recognized enzymatic activity but it is toxic for some helminths (Ackerman, S. J. et al., Am. J. Trop. Med. Hyg., 34:735-745 (1985)) and mammalian cells (Barker, R. L. et al., J. Clin. Invest. 88:798-805(1991)) in vitro. MBP is expressedprincipallyin bone-marroweosinophils, but it is also synthesized in basophils and placental trophoblast x-cells (Wasmoen, T. L. et al., J. Exp. Med., 170:2051-2063(1989)).
MBP stimulates the effector function of a wide variety of cells, namely MBP stimulates the noncytolytic release of histamine from human basophils and rat mass cells (O""Donnell, M. A. et al., J. Exp. Med. 157:1981 (1983)). MBP also stimulates neutrophils to release superoxide and ion and lysozyme, but not beta-glucuronidase or lactic dehydrogenase, and MBP enhances the expression of CR3 and P150, 95 by neutrophils. This indicates that MBP activates other cells associated with inflammation, such as basophils, platelets and neutrophils. The effector mechanisms may play a role in pathophysiology of various diseases where granule proteins are released. For example, MBP has been thought a cause for increased airway responsiveness, for example, bronchial asthma.
The effects of the natural killer cell activating factors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of proteins that influence biological activity, both normally and in diseased states. In particular, there is a need to isolate and characterize additional natural killer cell activating factors akin to known natural killer cell activating factors which may be employed, therefore, for preventing, ameliorating or correcting disfunctions or disease or augmenting positive natural actions of such receptors.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel NKAF II by homology between the amino acid sequence set out in FIG. 1 (SEQ ID NO:1 and 2) and known amino acid sequences of other proteins such as human eosinophil granule major basic protein.
It is a further object of the invention, moreover, to provide polynucleotides that encode NKAF II, particularly polynucleotidesthat encode the polypeptide herein designated NKAF II.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding human NKAF II in the sequence set out in FIG. 1 (SEQ ID NO:2).
In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97465.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding human NKAF II, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human NKAF II.
It also is an object of the invention to provide NKAF II polypeptides, particularly human NKAF II polypeptides, that inhibit the growth of leukemia cells, to treat viral infection, to augment the effects of natural killer protein to treat neoplasias such as tumors and cancers, to prevent inflammation, to treat parasitic infection, to regulate hematopoiesis, to prevent damage from superoxide radicals in the body, for example, tissue injury and aging and to enhance an immunological response.
In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as NKAF II as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
Among the particularly preferred embodiments of this aspect of the invention are variants of human NKAF II encoded by naturally occurring alleles of the human NKAF II gene.
It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned NKAF II polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derivedhuman NKAF II-encoding polynucleotide under conditions for expression of human NKAF II in the host and then recovering the expressed polypeptide.
In accordance with another object the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia, for, among other things: assessing NKAF II expression in cells by determining NKAF II polypeptides or NKAF II-encoding mRNA; assaying genetic variation and aberrations, such as defects, in NKAF II genes; and administering a NKAF II polypeptide or polynucleotide to an organism to augment NKAF II function or remediate NKAF II dysfunction.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided probes that hybridize to human NKAF II sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against NKAF II polypeptides. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for human NKAF II. In accordance with another aspect of the present invention, there are provided NKAF II agonists. Among preferred agonists are molecules that mimic NKAF II, that bind to NKAF II-binding molecules or receptor molecules, and that elicit or augment NKAF II-induced responses. Also among preferred agonists are molecules that interact with NKAF II or NKAF II polypeptides, or with other modulators of NKAF II activities, and thereby potentiate or augment an effect of NKAF II or more than one effect of NKAF II.
In accordance with yet another aspect of the present invention, there are provided NKAF II antagonists. Among preferred antagonists are those which mimic NKAF II so as to bind to NKAF II receptor or binding molecules but not elicit a NKAF II-induced response or more than one NKAF II-induced response. Also among preferred antagonists are molecules that bind to or interact with NKAF II so as to inhibit an effect of NKAF II or more than one effect of NKAF II or which prevent expression of NKAF II.
The agonists and antagonists may be used to mimic, augment or inhibit the action of NKAF II polypeptides. They may be used, for instance, to inhibit the action of such polypeptides, for example, to prevent allergic inflammation, hypersensitivity, bronchial asthma, eosinophilia, chronic urticaria, atopic dermatitis, Kimura""s disease and bone marrow transplant rejection.
In a further aspect of the invention there are provided compositions comprising a NKAF II polynucleotide or a NKAF II polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a NKAF II polynucleotide for expression of a NKAF II polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of NKAF II.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.